Project Summary: This project on group B Streptococcus (Streptococcus agalactiae) focuses on a class of key surface proteins called alpha-like proteins that are also found on a variety of other Gram-positive pathogens. These proteins, the prototype for which is the alpha C protein, are characterized by the presence of long tandem repeating subunits and are determinants of both virulence and immunity. Recent data show a role for the alpha-like proteins in binding to and invasion of human epithelial cells. Group B streptococci use the alpha C protein to interact with and enter host epithelial cells via host receptors that include glycosaminoglycans and integrins. These interactions may allow bacterial pathogens to invade host tissues. The long-term goal of this project is to understand how group B streptococcal alpha-like proteins function in virulence and immunity. The first specific aim examines mechanisms for regulation of expression of the alpha C protein by transcriptional factors outside its genetic locus, mechanisms we have shown to be involved in evasion of host immunity. Regulators of expression will be detected using DNA-affinity chromatography and through study of a library of transposon mutants. The second specific aim explores, at the molecular level, how the alpha C protein mediates the interaction of streptococci with host epithelial cells via recognition of glycosaminoglycans and integrins. Methods to be used include RNA interference, surface non-covalent affinity mass spectrometry, confocal microscopy and flow cytometry. These studies will lead to advances in the understanding of group B streptococcal pathogenesis and immunity, further the effort to employ these proteins in maternal vaccines for the prevention of group B streptococcal infection, and allow the development of inhibitors of bacterial invasion. Relevance: Group B streptococci are the leading cause of serious, often fatal, bacterial infection, including bloodstream infection, pneumonia, and meningitis in newborn babies. They are also a frequent cause of serious infection in women at childbirth and other adults with underlying illnesses such as cancer or diabetes. This project, which studies key proteins on the bacterial surface, will improve our understanding of how bacteria cause disease and allow the design of vaccines to protect pregnant women and their babies and medicines that will prevent and treat these devastating infections. [unreadable] [unreadable] [unreadable]